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Schedule

The method involves close collaboration with an investigator who will do all the mouse breeding to generate 4-5 plugged females by natural mating. The investigator should use the following breeding regime:

• You will generate 5 females plugged on the same day, on a day agreed to in advance with us. Because the matings are done without hormone stimulation, you will need 10 stud males and a large cohort of 25 young, sexually mature females (6-8 weeks). If you can identify estrus females, you can use a smaller number of males. The males should be experienced in mating but rested for a few days prior to attempting these critical matings.
• After coordinating with us, you will place 2-3 females with each male on the Sunday. On the Monday morning, you will check plugs. If one quarter of the females were in estrus, we would expect 5-6 females to have mated. Remove all the females and house the plugged females together. On Thursday, bring the plugged females up to your lab.

The females are brought up to the investigator’s lab and I do the dissection, flushing, embryo culture, embryo disaggregation, and culture of any resulting ES cell lines.

A rough outline of a single mating and derivation cycle is detailed. If a second round of mating is required, it is staggered by a week following the first round.

(Week 1)

Sunday: Investigator's lab sets-up matings

Monday: Check plugs. Set mated females aside. Unmated females can be used again (0.5 d.p.c.).

Tuesday: 1.5 d.p.c.

Wednesday: 2.5 d.p.c. Plate out PMEFs for embryo culture.

Thursday:

• Mice are brought up to investigator's laboratory (3.5 d.p.c.).
• I euthanize mice and dissect uteri.
• Return to transgenic lab and flush out 3.5 d.p.c. embryos.
• Embryos put into culture in individual 4-well PMEF using SR- medium (day 0).
• Depending on yield, decide if a second round of matings is required, based on goal of 25-40 embryos.

Friday: day 1.

Saturday: day 2.

(Week 2)

Sunday: day 3.

Monday: day 4.

Tuesday: day 5.

Wednesday: day 6--Primary embryo outgrowth disaggregation. (Hardest part). Disaggregations put into 4-well PMEFs with FBS-medium.

Thursday: day 7. Change medium to SR-medium.

Friday: day 8.

Saturday: day 9.

(Week 3)

Sunday: day 10.

Monday: day 11. Critical decision day. Is there anything worth passaging? Secondary passaging day for most outgrowths, using FBS- medium onto 4-well PMEFs. Each secondary becomes an individual cell line.

Tuesday: day 12. Switch to SR-medium and feed daily thereafter. Stragglers?

Wednesday: day 13. Stragglers? Secondaries from Monday (d.11) may be ready to passage soon. Begin passaging onto 35mm PMEFs as required. Passage in FBS-medium. Next day switch to SR-medium and feed daily thereafter.

Thursday: day 14. Passaging? Stragglers?

Friday: day 15. Passaging? Stragglers?

Saturday: day 16. Passaging? Stragglers?

(Week 4)

Sunday: day 17. Passaging? Stragglers?

Monday: day 18. Last chance for any stragglers. Passaging?

Freeze individual lines in vials as appropriate, removing aliquots for DNA. Evaluate DNA aliquots for likelihood of adaptation to growth on gelatinized plastic.

 

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